Fermentative preparation of l-isoleucine

ABSTRACT

1. A PROCESS FOR PREPARING L-ISOLEUCINE WHICH COMPRISES CULTIVATING AN ISOLEUCINE HYDROXAMATE-RESISTANT MUTANT OR AN ISOLEUCINE HYDROXAMATE- AND A-AMINOBUTYRIC ACID-RESISTANT MUTANT OF SERRATIA MARCESCENS IN A NUTRIENT MEDIUM UNDER AEROBIC CONDITIONS TO PRODUCE A FERMENTATION BROTH, AND RECOVERING ACCUMULATED L-ISOLEUCINE FROM THE FERMENTATION BROTH.

United States Patent 3,841,968 FERMENTATIVE PREPARATION OF L-ISOLEUCINEIchiro Chibata, Suita, Masahiko Kisumi, Kobe, Saburo Komatsubara, Kyoto,and Masaki Sugiura, Osaka, Japan, assignors to Tanabe Seiyaku Co., Ltd.

No Drawing. Continuation-impart of abandoned application Ser. No.201,400, Nov. 23, 1971. This application Aug. 21, 1972, Ser. No. 282,284

Int. Cl. C12d 13/06 U.S. Cl. 195-29 10 Claims ABSTRACT OF THE DISCLGSUREAn isoleucine hydroxamate-resistant mutant or an isoleucinehydroxamateand a-aminobutyric acid-resistant mutant of Serratiamarcescens is cultivated in an aqueous nutrient medium under aerobicconditions. 0.05 to 5 w./v. percent of L-threonine, L-homoserine orL-aspartic acid may be optionally added to the medium. The cultivationis preferably carried out at about 25 to 35 C. and at a pH of 6 to 9.L-isoleucine is recovered from the fermentation broth.

The present application is a continuation-in-part of application Ser.No. 201,400, filed Nov. 23, 1971, now abandoned.

This invention relates to the fermentative preparation of L-isoleucine.

L-isoleucine, one of the essential amino acids, has been employed as anactive ingredient of medicinal preparations and as a food additive.Known chemical synthesis of this amino acid is disadvantageous in thatfour stereoisomers of isoleucine are formed during reactions andcomplicated procedures are required to separate biologically activeL-isoleucine from the mixture of the isomers. It has been recognized,therefore, that a fermentative method is most advantageous and practicalin the industrial preparation of L-isoleucine.

There have been known three methods in the fermentative preparation ofL-isoleucine, but these methods necessarily require the use of aprecursor in the fermentation. Known methods may be divided into twogroups. One is the method of Japanese patent publication No. 6,593/ 1963wherein cultivating a strain of Pseudomonas or Serratia genus in anutrient medium containing D- threonine is employed. The other is themethods of Japanese patent publication Nos. 18,511/ 1961 and 5,167/ 1962wherein cultivating a strain of Bacillus or Pseudornonas genus in anutrient medium containing DL-OL- aminobutyric acid is employed.Moreover, L-threonine can not be used as the precursor in the method ofthe former group. (Jap. Pat. Pub. No. 6,593/1963.)

As a result of, various investigations, we have found that a mutant ofSerratia marcescens resistant to isoleucine hydroxamate or resistant toboth isoleucine hydroxamate and a-aminobutyric acid has an excellentproductivity of L-isoleucine from a normal source of carbon.Additionally, we have also found that the L-isoleucine productivity ofthe mutant can be remarkably enhanced by carrying out the fermentationin the presence of L- threonine, L-homoserine or L-aspartic acid.

According to the present invention, L-isoleucine can be prepared bycultivating an isoleucine hydroxamateresistant mutant or an isoleucinehydroxamateand otaminobutyric acid-resistant mutant of Serratiamarcescens in a nutrient medium under aerobic conditions.

The isoleucine hydroxamate-resistant mutant of the present invention maybe obtained by ultraviolet irradiation of a wild type strain of Serratiamarcescens or by treating saidwild type strain with a mutagen. Forinstance, a Wild type strain of Serratia marcescens is treated 3,841,968Patented 'Oct. 1 5, 1974 Too with N-methyl-N-nitro-N-nitrosoguanidineand then cultured at 30 C. for 3 to 5 days on agar plates containing thefollowing compositions: K HPO 0.7 w./v. percent; KH PO 0.3 w./v.percent; MgSO 7H O, 0.01 w./v. percent; (NH SO 0.1 w./v. percent;glucose, 0.5 w./v. percent; L-isoleucine hydroxamate, 1 mg./ml. Theisoleucine hydroxamate-resistant mutant of Serratia marcescens may beisolated as large colonies. Alternatively, the isoleucine hydroxamateanda-aminobutyric acidresistant mutant of the present invention may beisolated as large colonies when the isoleucine hydroxamate-resistantmutant is cultured at 30 C. for 2 to 3 days on the agar platescontaining the same ingredients as mentioned above except that 10mg./ml. of a-aminobutyric acid is employed instead of isoleucinehydroxamate. A viable culture of a isoleucine hydroxamate-resistantmutant of Serratia marcescens has been deposited with the American TypeCulture Collection, 12301 Parklawn Drive, Rockville, Md. 20852(hereinafter referred to as ATCC) under No. 21,741, and a viable cultureof a isoleucine hydroxamateand a-aminobutyric acid-resistant mutant ofSerratia marcescens has also deposited at the same depository under ATCCNo. 21,740 and ATCC No. 21,810.

The fermentation of the mutant of Serratia marcescens can beaccomplished by either shaking cultivation or submerged fermentationunder aerobic conditions. The fermentation may be preferably carried outat 25 to 37 C. and at a pH of 6 to 9. Calcium carbonate and ammonia maybe employed for adjustment of the pH of the medium. The fermentationmedium contains a source of carbon, a source of nitrogen and otherelements. Suitable sources of carbon for the fermentation includeglucose, starch hydrolysate, fumaric acid, citric acid and glycerol.Examples of suitable sources of nitrogen are urea, ammonium salts oforganic acids (e.g., ammonium acetate, ammonium oxalate) and ammoniumsalts of inorganic acids (e.g., ammonium sulfate, ammonium nitrate).Preferred amount of the source of carbon and the source of nitrogen inthe medium are respectively within the range of 2 to 15 w./v. percentand 0.5 to 2 w./v. percent. Furthermore, organic nutrients (e.g., cornsteep liquor, peptone, yeast extracts) and/or inorganic elements (e.g.,potassium phosphate, magnesium sulfate) may be added to the medium.

In carrying out the fermentation of the instant invention, L-isoleucineproductivity of the above-mentioned mutants may be further enhanced byaddition of about 0.05 to 5 w./v. of L-threonine, L-homoserine orL-aspartic acid. The fermentation of the invention can be accomplishedin about 24 to 96 hours. L-isoleucine is accumulated in the fermentationbroth.

After the fermentation is completed, cells and other solid culturecompositions are removed from the fermentation broth by conventionalprocedures such as by heating, followed by filtration or centrifugation.Known procedures may be employed in the recovery and/0r purification ofL-isoleucine from the filtrate or the supernatant solution. Forinstance, the filtered fermentation broth is passed through or treatedwith a strong cation exchange resin. Then, the resin is eluted with adilute alkaline solution such as aqueous ammonia. The eluates containingL-isoleucine are combined and concentrated. An alkanol such as methanoland ethanol is added to the concentrated solution. The precipitatedcrystals are recrystallized from an aqueous alkanol such as aqueousmetha'nol and aqueous ethanol to yield pure crystals of L-isoleucine.

Practical and presently-preferred embodiment of this invention areillustratively shown in the following examples.

3 EXPERIMENT Serratia marcescens OUT (Faculty of Engineering, OsakaUniversity) 8259, the isoleucine hydroxamate-resistant mutant ATCC No.21,741of Serratia marcescens and the isoleucine hydroxamateanda-aminobutyric acid-resistant mutant ATCC No. 21,740 of Serratiamarcescens are respectively cultivated for 48 hours at 30 C. in anutrient medium (pH 7.0) (Ingredients: glucose, 2 w./v. percent;dextrine, 7 W./V. percent; urea, 0.5 w./v. percent; corn steep liquor,0.35 w./v. percent; dibasic potassium phosphate, 0.1 w./v. percent;magnesium sulfate, 0.05 w./v. percent; calcium carbonate, 2 w./v.percent) or in a nutrient medium (pH 7.0) containing the above-mentionedingredients and 1.0 W./V. percent of L- threonine, L-homeserine orL-aspartic acid. The amount of L-isoleucine accumulated in the mediumare shown in the following Table.

Q An aqueous nutrient medium comprising the following ingredients isprepared:

W./v. percent L-threonine 2 Glucose 2 Dextrin 10 Urea 0.5 Di-basicpotassium phosphate 0.1 Magnesium sulfate 0.05 Calcium carbonate 2 Theabove medium is adjusted to pH 7.0. ml. of the medium are charged into a500 ml. shaking flask and its contents are sterilized by autoclaving. Aloopful of the isoleucine hydroxamate-resistant mutant ATCC No. 21,741of Serratia marcescens is inoculated aseptically into the medium. Then,the medium is cultivated for 48 hours at 30 C. under shaking. Thefermentation medium thus obtained contains 7 mg./ml. of L-isoleucine.

1000 ml. of the fermentation medium are heated at 100 C. for 10 minutesand then filtered. The filtrate is introduced into a column (3 cm. x 45cm.) of strong cation exchange resin (H-form) manufactured by Rohm &Haas Company under the trade name Amberlite IR- 120. After washing withwater, the column is eluted with 5% aqueous ammonia. The fractionscontaining L-isoleucine are collected and concentrated under reducedpressure to about 80 ml. 100 ml. of aqueous methanol are added to thesolution. The precipitating crystals are collected by filtration andthen recrystallized from aqueous methanol. 5 g. of L-isoleucine areobtained.

[a] =+40.1 (C: 1,6N-hydrochloric acid).

Example 2 An aqueous nutrient medium comprising the followingingredients is prepared:

W./v. percent Glucose 5 Ammonium fumarate 2 Urea 0.5 Dibasic potassiumphosphate 0.1 Magnesium sulfate 0.05 Calcium carbonate 2 EXAMPLE 3 15ml. of a nutrient medium comprising the same ingredients as described inExample 1 except that 4 w./v. percent of DL-threonine instead ofL-threonine is prepared. After sterilization, a loopful of theisoleucine hydroxamate-resistant mutant ATCC No. 21,741 of Serratiamarcescens is inoculated aseptically into the medium. Then, the mediumis cultivated for 72 hours at 30 C. under shaking. The fermentationmedium thus obtained contains 13 mg./ml. of L-isoleucine.

EXAMPLE 4 15 ml. of a nutrient medium comprising the same ingredients asdescribed in Example 3 is prepared. After sterilization, a loopful ofthe isoleucine hydroxamateand ot-aminobutyric acid-resistant mutant ATCCNo. 21,- 740 of Serrazia marcescens is inoculated aseptically into themedium. Then, the medium is cultivated in the same manner as describedin Example 1. The fermentation medium thus obtained contains 25 mg./ml.of L-isoleucine.

EXAMPLE 5 An aqueous nutrient medium comprising the followingingredients is prepared:

W./v. percent L-homoserine 1 Sorbitol 8 Powdered corn steep liquor 0.35Urea 0.5 Dibasic potassium phosphate 0.1 Magnesium sulfate 0.05 Calciumcarbonate 2 The above medium is adjusted to pH 7.0. 15 ml. of the mediumis charged into a 500 ml. shaking flask and its contents are sterilizedby autoclaving. A loopful of the isoleucine hydroxamateandu-aminobutyric acid-resistant mutant ATCC No. 21,740 of Serratiamarcescens is inoculated aseptically into the medium. Then, the mediumis cultivated in the same manner as described in Example 1. Thefermentation medium thus obtained contains 4.5 mg./ ml. of L-isoleucine.

EXAMPLE 6 An aqueous nutrient medium comprising the followingingredients is prepared:

W./v. percent Glucose Glycerol 1 Powdered corn steep liquor 0.35 Urea0.5 Dibasic potassium phosphate 0.1 Magnesium sulfate 0.05 Calciumcarbonate 2 The above medium is adjusted to pH 7.0. 15 ml. of the mediumare charged into a 500 ml. shaking flask and its contents are sterilizedby autoclaving. A loopful of the isoleucine hydroxamateandu-aminobutyric acid-resistant mutant ATCC No. 21,740 of Serratiamarcescens is inoculated aseptically into the medium. Then, the mediumis cultivated for 72 hours at 30 C. under shaking. The fermentationmedium thus obtained contains 2.0 mg./ ml. of L-isoleucine.

EXAMPLE 7 EXAMPLE 8 An aqueous nutrient medium comprising the followingingredients is prepared:

W./v. percent Glucose Dextrin Urea 1.0 Secondary potassium phosphate -40.1 Magnesium sulfate 0.05 Calcium carbonate 2 The above medium isadjusted to pH 7.0. 15 ml. of the medium are charged into a 500 ml.shaking flask and its contents are sterilized by autoclaving. A loopfulof the isoleucine hydroxamateand a-aminobutyric acid-resistant mutantATCC 21,810 of Serratia marcescens is inoculated aseptically into themedium. Then, the medium is cultivated at 30 C. for 48 hours undershaking. The fermentation medium thus obtained contains 6.5 mg./ml. ofL-isoleucine.

EXAMPLE 9 An aqueous nutrient medium comprising the followingingredients is prepared:

W./v. percent 4 Dextrin 10 Urea 1 Secondary potassium phosphate 0.1Magnesium sulfate 0.05 Calcium carbonate 2 The above medium is adjustedto pH 7.0. ml. of the medium are charged into a 500 ml. shaking flaskand its contents are sterilized by autoclaving. A loopful of theisoleucine hydroxamateand ot-aminobutyric acid-resistant mutant ATCC21,740 of Serratia marcescens is inoculated aseptically into the medium.Then, the medium is cu1ti vated at 30 C. for 48 hours under shaking. Thefermentation medium thus obtained contains 3.8 mg./ml. of L-isoleucine.

Glucose What We claim is:

1. A process for preparing L-isoleucine which comprises cultivating anisoleucine hydroxamate-resistant mutant or an isoleucine hydroxamateanda-aminobutyric acid-resistant mutant of Serratia marcescens in anutrient medium under aerobic conditions to produce a fermentationbroth, and recovering accumulated L-isoleucine from the fermentationbroth.

2. The process according to claim 1, wherein the mutant is Serratiamarcescens AT CC No. 21,740.

3. The process according to claim 1, wherein the mutant is Serratz'amarcescens ATCC No. 21,741.

4. The process according to claim 1, wherein the mutant is Serratiamarcescens ATCC No. 21,810.

5. The process according to claim 1, wherein the cultivation is carriedout in the presence of 0.05 to 5 w./v. percent of L-threonine,L-homoserine or L-aspartic acid.

6. The process according to claim 1, wherein the cultivation is carriedout at about 25 to about 35 C.

7. The process according to claim 1, wherein the cultivation is carriedout at a pH of 6 to 9.

8. The process according to claim 1, wherein the cultivation is carriedout in the presence of 0.05 to 5 w./v. percent, based on the medium, ofL-threonine, L-homoserine or L-aspartic acid at about 25 to about 35 C.andatapH of6to9.

9. A process for preparing L-isoleucine which comprises cultivatingisoleucine hydroxamate-resistant mutant ATCC No. 21,741 of Serratiamarcescens in a nutrient medium containing 0.05 to 5 w./v. percent ofL-threonine, L-homoserine or L-aspartic acid under aerobic conditions atabout 25 to about 35 C. and at a pH of 6 to 9 to produce a fermentationbroth, and recovering accumulated L-isolucine from the fermentationbroth.

10. A process for preparing L-isoleucine which comprises cultivatingisoleucine hydroxamateand a-aminobutyric acid-resistant mutant ATCC No.21,740 of Serratia marcescens in a nutrient medium containing 0.05 to 5w./v. percent, based on the medium, of L-threonine, L-homoserine orL-aspartic acid under aerobic conditions at about 25 to about 35 C. andat a pH of 6 to 9 to produce a fermentation broth, and recoveringaccumulated L-isoleucine from the fermentation broth.

References Cited UNITED STATES PATENTS 3,086,918 4/1963 Chibata et a1.-47

ALVIN E. TANENHOLTZ, Primary Examiner US. Cl. X.R. 19530, 47, 112

1. A PROCESS FOR PREPARING L-ISOLEUCINE WHICH COMPRISES CULTIVATING ANISOLEUCINE HYDROXAMATE-RESISTANT MUTANT OR AN ISOLEUCINE HYDROXAMATE-AND A-AMINOBUTYRIC ACID-RESISTANT MUTANT OF SERRATIA MARCESCENS IN ANUTRIENT MEDIUM UNDER AEROBIC CONDITIONS TO PRODUCE A FERMENTATIONBROTH, AND RECOVERING ACCUMULATED L-ISOLEUCINE FROM THE FERMENTATIONBROTH.